Antibodies specific to pyroglutamated Aβ

ABSTRACT

The present invention relates to antibodies specific for pyroglutamated Aβ, as well as their use in the treatment of Alzheimer&#39;s disease and as use in diagnostic methods or as diagnostic imaging ligands. Further, is provided pyroglutamated N-terminal fragments of murine or human Aβ to generate antibodies and for use in therapeutic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 14/009,492, (filed on Oct. 11, 2013;patented), which application is a § 371 application of US PatentApplication Serial No. PCT/EP2012/055598 (filed on Mar. 29, 2012; nowlapsed), which claims priority to U.S. Patent Application Ser. No.61/473,178 (filed on Apr. 8, 2011) and and Denmark ApplicationPA201100275 having a filing date of Apr. 8, 2011, each of whichapplications is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies specific for pyroglutamatedAβ, as well as their use in the treatment of Alzheimer's disease and asuse in diagnostic methods or as diagnostic imaging ligands. Further, isprovided pyroglutamated N-terminal fragments of murine or human Aβ togenerate antibodies and for use in therapeutic purposes.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(file name: 1275_0020C2_ST25.txt, created on Oct. 6, 2016, and having asize of 28,472 bytes), which file is herein incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

Alzheimer disease (AD) is characterised by a progressive dementiaoccurring in mid or late life. The first events leading to AD arebelieved to occur some 10 to 20 years before the cognitive symptomsappear, and evidence suggests that a key event in the pathology of AD isthe deposit of extracellular aggregated amyloid β (Aβ) plaques in thebrain. Aβ is generated by proteolytic cleavage from the amyloidprecursor protein (APP) which is a large transmembrane protein with asuggested neutrophic function. The main Aβ variants observed in thehuman brain are Aβ 1-40 and Aβ 1-42, but also truncated N-terminalvariants and other modified species thereof are observed in the plaques.

Immunotherapy against the Aβ plaques has been shown to disrupt the Aβaggregates and promote the clearance of plaques in the brain. Bothactive immunogenic approaches with Aβ or fragments thereof as well aspassive immunisation using anti-Aβ antibodies have proven effective indifferent animal models. Vaccination of humans with Aβ was shown toprevent the development of Aβ plaques and reduce the Aβ burden inpatients in a clinical study, however the study was stopped due toinflammation in the brain of some of the patients. An immunogenicstrategy to reduce the amyloid plaques thus relies on a delicate balanceof activating the immune system but still avoiding an uncontrollableinflammation to occur (Monsonego et al (2003) J clin invest 112,415-422). This requires a precise knowledge of the chemical nature ofinter alia the nature of the deposit. In an attempt to understand theexact composition of the amyloid plaques one of the most abundantN-truncated Aβ peptides identified are carrying a pyroglutamate atposition 3 (“AβpE3,” N-terminally truncated Aβ starting withpyroglutamate) (Saido et al. (1996) Neuron 14, 457-466). Thesepyroglutamated peptides have been shown to accumulate in plaques as wellas around the blood vessels in AD, and due to its hydrophobic potentialit has been shown that these peptides increase the aggregation. A recenttransgenic mouse model expressing AβpE3-42 in neurons demonstrates thatthis peptide is neurotoxic in vivo and leads to loss of neurons (Wirthset al. (2009) Acta Neuropathol 118, 487-496).

Antibodies with specificities against the N-terminal pyroglutamate arebelieved to be advantageous because of their specificity towards onlythe pathogenic pyroglutamate N-terminal species of Aβ, thereby leavingAPP or other cross reacting species untouched. It is thus envisaged thatthe risk of uncontrollable cerebral inflammation will be reducedcompared to antibodies directed to non-pyroglutamated Aβ or fragmentsthereof.

Antibodies targeting AβpE3 peptides and aggregates have been made (Aceroet al (2009) J Neuroimmunol 213, 39-46; Saido et al. (1996) Neuron 14,457-466; and U.S. Pat. No. 7,122,374) and identification of various AβpEfragments have been hypothesized as epitopes (WO2004/013172;WO2010/009987; WO2010/129276; and Wirths et al. (2010) J Biol Chem285(53), 41517-24, Epub 2010 Oct. 22).

The present invention provides two highly specific monoclonal antibodiesdirected against human AβpE3 (5C9 and 2E83, respectively) as well as twohighly specific monoclonal antibodies directed against murine AβpE3 (2E4and 1G11, respectively).

SUMMARY OF THE INVENTION

The invention relates to an antibody, in particular an isolatedantibody, or a fragment thereof, specific for human AβpE3 which antibodycomprises, or consists, of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising        GYTFTDYYX₁N (SEQ ID NO:56),    -   a heavy chain variable region (V_(H)) CDR2 comprising        WX₂X₃PGSGNX₄KYNEKFKG (SEQ ID NO:57),    -   a heavy chain variable region (V_(H)) CDR3 comprising EGX₅X₆X₇Y        (SEQ ID NO:58),    -   a light chain variable region (V_(L)) CDR1 comprising        KSSQSLLX₈SNGX₉X₁₀YLN (SEQ ID NO:59),    -   a light chain variable region (V_(L)) CDR2 comprising X₁₁VSKLDS        (SEQ ID NO:60), and    -   a light chain variable region (V_(L)) CDR3 comprising        VQGTHX₁₂PFT (SEQ ID NO:61);    -   wherein X₁ to X₁₂ symbolizes a natural amino acid.

The invention further relates to an antibody, in particular an isolatedantibody, or a fragment thereof, specific for murine AβpE3 whichantibody comprises, or consists, of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising        GX₁TLX₂DAWMX₃ (SEQ ID NO:62),    -   a heavy chain variable region (V_(H)) CDR2 comprising        EIRX₄KAX₅X₆HATX₇YAESVKG (SEQ ID NO:63),    -   a heavy chain variable region (V_(H)) CDR3 comprising HX₈X₉ (SEQ        ID NO:64),    -   a light chain variable region (V_(L)) CDR1 comprising        X₁₀ASQGIX₁₁X₁₂X₁₃X₁₄G (SEQ ID NO:65),    -   a light chain variable region (V_(L)) CDR2 comprising HGTKLED        (SEQ ID NO:36), and    -   a light chain variable region (V_(L)) CDR3 comprising        VQYX₁₅QFPYT (SEQ ID NO:66)    -   wherein X₁ to X₁₅ symbolizes a natural amino acid.

In further embodiments the invention provides four specific monoclonalantibodies as set forth in the claims, as well as their use in thetreatment of Alzheimer's disease and as use in diagnostic methods or asdiagnostic imaging ligands. The pyroglutamated N-terminal 3-11 aminoacids of murine or human Aβ is also provided for the generation ofantibodies or for use in pharmaceutical compositions.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B. Biacore binding of pE3-specific monoclonal antibodies(MABs) to human AβpE3 (FIG. 1A) and murine AβpE3 (FIG. 1B). Data for twomonoclonal antibodies highly specific for human AβpE3, LuAb1h and LuAb2h(which are the same as 5C9 and 2E83, respectively), and two monoclonalantibodies highly specific for murine AβpE3, exemplified by LuAb1m andLuAb2m is shown.

FIGS. 2A and 2B. Shows the Biacore binding curves to human AβpE3 (FIG.2A) and murine AβpE3 (FIG. 2B) peptides for the antibodies in FIG.1A-1B.

FIGS. 3A and 3B. Micrographs showing immunoreactivity of Abeta andpE3-Abeta antibodies in the frontal cortex from an Alzheimer brain (FIG.3A) and in the hippocampal region of an APP/PS1 transgenic mouse (FIG.3B). The antibodies used are 6E10, which is specific for Aβ, 2E8 and 5C9which are specific for human AβpE.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “pyroglutamated Aβ”, and “AβpE” are used interchangeablyherein, and, unless specified otherwise, include any variants, isoformsand species homologs of pyroglutamated A. For example, AβpE3 refers todifferent Aβ variants, including, but not limited to the fragmentsAβ3-42, Aβ3-40 or Aβ3-11, which are pyroglutamated at the N-terminal endspecifically at the position 3 glutamic acid.

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,which may all four be inter-connected by disulfide bonds. The structureof immunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as V_(H) or VH) and a heavychain constant region. The heavy chain constant region typically iscomprised of three domains, C_(H)1, C_(H)2, and C_(H)3. Each light chaintypically is comprised of a light chain variable region (abbreviatedherein as V_(L) or VL) and a light chain constant region. The lightchain constant region typically is comprised of one domain, C_(L). TheV_(H) and V_(L) regions may be further subdivided into regions ofhypervariability (or hypervariable regions which may be hypervariable insequence and/or form of structurally defined loops), also termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FRs). Each V_(H) andV_(L) is typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxyterminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196,901-917 (1987)). Typically, the numbering of amino acid residues in thisregion is according to IMGT, Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991).

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule or according to some embodiments of theinvention may be a fragment of an immunoglobulin molecule, or aderivative of either thereof, which has the ability to specifically bindto an antigen. The variable regions of the heavy and light chains of theimmunoglobulin molecule contain a binding domain that interacts with anantigen as outlined above under “immunoglobulin”. An anti-AβpE3 antibodymay also be a bispecific antibody, diabody, or similar molecule (see forinstance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies).Further, it has been shown that the antigen-binding function of anantibody may be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term “antibody”include (i) a Fab′ or Fab fragment, a monovalent fragment consisting ofthe V_(L), V_(N), C_(L) and C_(H)1 domains, or a monovalent antibody asdescribed in WO2007059782; (ii) F(ab′)₂ fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting essentially of the V_(H) andC_(H)1 domains; (iv) a Fv fragment consisting essentially of a V_(L) andV_(H) domains, (v) a dAb fragment (Ward et al., Nature 341, 544-546(1989)), which consists essentially of a V_(H) domain and also calleddomain antibodies (Holt et al; Trends Biotechnol. 2003 November;2i(II):484-90); (vi) camelid or nanobodies (Revets et al; Expert OpinBiol Ther. 2005 January; 5_(I): I II-24) and (vii) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they may be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe V_(L) and V_(H) regions pair to form monovalent molecules (known assingle chain antibodies or single chain Fv (scFv), see for instance Birdet al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85,5879-5883 (1988)). Such single chain antibodies are encompassed withinthe term antibody unless otherwise noted or clearly indicated bycontext. Although such fragments are generally included within themeaning of antibody, they collectively, and each independently, areunique features of the present invention, exhibiting differentbiological properties and utility. These and other useful antibodyfragments in the context of the present invention are discussed furtherherein. It also should be understood that the term antibody, unlessspecified otherwise, also includes polyclonal antibodies, monoclonalantibodies (MAbs), antibody-like polypeptides, such as chimericantibodies and humanized antibodies, and antibody fragments retainingthe ability to specifically bind to the antigen (antigen-bindingfragments) provided by any known technique, such as enzymatic cleavage,peptide synthesis, and recombinant techniques. An antibody as generatedcan possess any isotype.

An “anti-AβpE3 antibody” is an antibody as described above, which bindsspecifically to variant fragments of Aβ, including, but not limited to,pyroglutamated N-terminal truncated forms such as the fragments Aβ3-42,3-40 or 3-11, which are pyroglutamated in the N-terminal end at position3.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or during gene rearrangement or bysomatic mutation in vivo).

The term “humanized antibody”, as used herein, is intended to includeantibodies in which CDR sequences are derived from the germline ofanother mammalian species, such as a mouse, and have been grafted ontohuman framework sequences. Humanized monoclonal antibodies may begenerated by a hybridoma which includes a B lymphocyte cell obtainedfrom a transgenic or transchromosomal nonhuman animal, such as atransgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene, fused to an immortalized cell.For example, when non-human antibodies are prepared with respect to aparticular antigen, the variable regions can be “reshaped” or humanized”by grafting CDRs derived from nonhuman antibody on the FRs present inthe human antibody to be modified. Application of this approach has beenreported by Sato, K. et al. Cancer Research 53:851-856 (1993);Riechmann, L., et al., Nature 332:323-327 (1988); Verhoeyen, M., et al.,Science 239:1534-1536 (1988); Kettleborough, C. A., et al., ProteinEngineering 4:773-3783 (1991); Maeda, H., et al., Human AntibodiesHybridoma 2:124-134 (1991); Gorman, S. D., et al., Proc Natl Acad SciUSA 88:4181-4185 (1991); Tempest, P. R., et al., Bio/Technology9:266-271 (1991); Co, M. S., et al., Proc Natl Acad Sci USA 88:2869-2873(1991); Carter, P., et al., Proc Natl Acad Sci USA 89:4285-4289 (1992);and Co, M. S. et al., J Immunol 148:1149-1154 (1992). In someembodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, or six) which are alteredwith respect to the original antibody, which are also termed one or moreCDRs “derived from” one or more CDRs from the original antibody.Humanized antibodies may refer to chimeric molecules prepared usingrecombinant techniques.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “mouse or murine monoclonal antibody” refers toantibodies displaying a single binding specificity which have variableand constant regions derived from murine or mouse germlineimmunoglobulin sequences.

As used herein, the term “binding” in the context of the binding of anantibody to a predetermined antigen typically refers to binding with anaffinity corresponding to a K_(D) of about 10⁻⁷ M or less, such as about10⁻⁸ M or less, such as about 10⁻⁹ M or less when determined by forinstance surface plasmon resonance (SPR) technology in a BIAcore 3000instrument using the antigen as the ligand and the antibody as theanalyte, and binds to the predetermined antigen with an affinitycorresponding to a K_(D) that is at least ten-fold lower, such as atleast 100 fold lower, for instance at least 1,000 fold lower, such as atleast 10,000 fold lower, for instance at least 100,000 fold lower thanits affinity for binding to a non-specific antigen (e.g., BSA, casein)other than the predetermined antigen or a closely-related antigen. Theamount with which the affinity is lower is dependent on the K_(D) of theantibody, so that when the K_(D) of the antibody is very low (that is,the antibody is highly specific), then the amount with which theaffinity for the antigen is lower than the affinity for a non-specificantigen may be at least 10,000 fold.

The term “k_(d)” (sec⁻¹ or 1/s), as used herein, refers to thedissociation rate constant of a particular antibody-antigen interaction.Said value is also referred to as the k_(off) value.

The term “k_(a)” (M⁻¹× sec⁻¹ or 1/M), as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction.

The term “K_(D)” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction.

The term “K_(A)” (M⁻¹ or 1/M), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the k_(a) by the k_(d).

The present invention also provides antibodies comprising functionalvariants of the V_(L) region, V_(H) region, or one or more CDRs of theantibodies of the examples. A functional variant of a V_(L), V_(H), orCDR used in the context of an anti-AβpE3 antibody still allows theantibody to retain at least a substantial proportion (at least about80%, 90%, 95% or more) of the affinity and/or thespecificity/selectivity of the parent antibody and in some cases such ananti-AβpE3 antibody may be associated with greater affinity, selectivityand/or specificity than the parent antibody. Such functional variantstypically retain significant amino acid sequence identity to the parentantibody. The percent identity between two amino acid sequences is afunction of the number of identical positions shared by the sequences(i.e., % homology=# of identical positions/total # of positions×100),taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences may be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The sequence of CDR variants may differ from the sequence of the CDR ofthe parent antibody sequences through substitutions; for instancesubstituted at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 ofthe amino acid residues. According to an embodiment of the invention itis furthermore envisaged that the amino acids in the CDR regions may besubstituted with conservative substitutions, as defined in the below 3tables. For example, the acidic residue Asp can be substituted with Gluwithout substantially affecting the binding characteristic of theantibody.

In the context of the present invention, conservative substitutions maybe defined by substitutions within the classes of amino acids reflectedin one or more of the following three tables:

Amino Acid Residue Classes for Conservative Substitutions:

Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg (R), andHis (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), andGln (Q) Aliphatic Uncharged Residues Gly (G), Ala (A), Val (V), Leu (L),and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P)Aromatic Residues Phe (F), Tyr (Y), and Trp (W)Alternative Conservative Amino Acid Residue Substitution Classes:

1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y WAlternative Physical and Functional Classifications of Amino AcidResidues:

Alcohol group-containing residues S and T Aliphatic residues I, L, V,and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, 1, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turnformation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q,T, K, S, G, P, D, E, and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W. H. Freeman and Company.

In one embodiment of the present invention, conservation in terms ofhydropathic/hydrophilic properties and residue weight/size also issubstantially retained in a variant CDR as compared to a CDR of anantibody of the examples (e.g., the weight class, hydropathic score, orboth of the sequences are at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or more retained). For example,conservative residue substitutions may also or alternatively be based onthe replacement of strong or weak based weight based conservationgroups, which are known in the art.

The retention of similar residues may also or alternatively be measuredby a similarity score, as determined by use of a BLAST program (e.g.,BLAST 2.2.8 available through the NCBI using standard settings BLOSUM62,Open Gap=I I and Extended Gap=I). Suitable variants typically exhibit atleast about 80%, at least about 90%, at least about 95%, or moresimilarity to the parent peptide.

As used herein, “isotype” refers to the immunoglobulin class (forinstance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encodedby heavy chain constant region genes.

The term “epitope” means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and nonconformationalepitopes are distinguished in that the binding to the former, but notthe latter, is lost in the presence of denaturing solvents. The epitopemay comprise amino acid residues directly involved in the binding (alsocalled immunodominant component of the epitope) and other amino acidresidues, which are not directly involved in the binding, such as aminoacid residues which are effectively blocked by the specifically antigenbinding peptide (in other words, the amino acid residue is within thefootprint of the specifically antigen binding peptide).

As used herein, a humanized antibody is “derived from” a particulargermline sequence if the antibody is obtained from a system using humanimmunoglobulin sequences, for instance by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library, and wherein the selected human antibody Vdomain sequence is at least 90%, such as at least 95%, for instance atleast 96%, such as at least 97%, for instance at least 98%, or such asat least 99% identical in amino acid V domain sequence to the amino acidsequence encoded by the germline immunoglobulin gene.

Typically, outside the heavy chain, a human antibody derived from aparticular human germline sequence will display no more than 20 aminoacid differences, e.g. no more than 10 amino acid differences, such asno more than 9, 8, 7, 6 or 5, for instance no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. For example, a transgenic mouse can have a humanlight chain transgene and either a human heavy chain transgene or humanheavy chain transchromosome, such that the mouse produces humananti-AβpE3 antibody when immunized with AβpE3 antigen and/or cellsexpressing AβpE3. The human heavy chain transgene may be integrated intothe chromosomal DNA of the mouse, as is the case for transgenic mice,for instance HuMAb mice, such as HCo7 or HCol2 mice, or the human heavychain transgene may be maintained extrachromosomally, as is the case fortranschromosomal KM mice as described in WO02/43478. Such transgenic andtranschromosomal mice (collectively referred to herein as “transgenicmice”) are capable of producing multiple isotypes of human monoclonalantibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) byundergoing V-D-J recombination and isotype switching.

Transgenic, nonhuman animal can also be used for production ofantibodies against a specific antigen by introducing genes encoding suchspecific antibody, for example by operatively linking the genes to agene which is expressed in the milk of the animal.

The term “treatment” or “treating” as used herein means ameliorating,slowing or reversing the progress or severity of a disease or disorder,or ameliorating, slowing or reversing one or more symptoms or sideeffects of such disease or disorder. For purposes of this invention,“treatment” or “treating” further means an approach for obtainingbeneficial or desired clinical results, where “beneficial or desiredclinical results” include, without limitation, alleviation of a symptom,diminishment of the extent of a disorder or disease, stabilized (i.e.,not worsening) disease or disorder state, delay or slowing of theprogression a disease or disorder state, amelioration or palliation of adisease or disorder state, and remission of a disease or disorder,whether partial or total, detectable or undetectable.

An “effective amount”, when applied to an antibody of the invention,refers to an amount sufficient, at dosages and for periods of timenecessary, to achieve an intended biological effect or a desiredtherapeutic result including, without limitation, clinical results. Thephrase “therapeutically effective amount” when applied to an antibody ofthe invention is intended to denote an amount of the antibody that issufficient to ameliorate, palliate, stabilize, reverse, slow or delaythe progression of a disorder or disease state, or of a symptom of thedisorder or disease. In an embodiment, the method of the presentinvention provides for administration of the antibody in combinationswith other compounds. In such instances, the “effective amount” is theamount of the combination sufficient to cause the intended biologicaleffect.

A therapeutically effective amount of an anti-AβpE3 antibody may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the anti-AβpE3 antibody to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the antibody orantibody portion are outweighed by the therapeutically beneficialeffects.

FURTHER ASPECTS OF THE INVENTION

The present invention encompasses the production of novel monoclonalantibodies with specificities for human AβpE3, particularly humanAβpE3-11 peptide, relative to murine AβpE3-11 peptide. The inventionprovides a method for producing anti-AβpE3 monoclonal antibodies thatspecifically bind AβpE3 peptides, particularly human AβpE3-11, with agreater affinity than said monoclonal antibodies bind murine AβpE3-11,said method comprising: (a) immunizing one or more mice with purifiedhuman AβpE3-11-C peptide; (b) producing hybridoma cell lines from spleencells of said one or more mice; (c) screening said hybridoma cell linesfor one or more hybridoma cell lines that produce antibodies thatspecifically bind human AβpE3-11, with a greater affinity than saidmonoclonal antibodies bind murine AβpE3-11.

Comparison between antibodies specific for human and murine AβpE3,particularly the AβpE3-11 peptide, revealed substantial conservationamong several of the amino acids in the CDR regions as well as positionsthat allows different types of amino acid replacements.

Specific Clone for V_(H) CDR1 V_(H) CDR2 V_(H) CDR3 5C9 Human GYTFTDYYLNWIYPGSGNVKYNEKFKG EGLIVY AβpE3-11 (SEQ ID NO: 12) (SEQ ID NO: 13)(SEQ ID NO: 14) 2E8 Human GYTFTDYYIN WLNPGSGNTKYNEKFKG EGIPDY AβpE3-11(SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO: 20) Difference GYTFTDYYXNWXXPGSGNXKYNEKFKG EGXXXY (SEQ ID NO: 44) (SEQ ID NO: 45) (SEQ ID NO: 46)V_(L) CDR1 V_(L) CDR2 V_(L) CDR3 5C9 Human KSSQSLLHSNGESYLN AVSKLDSVQGTHFPFT AβpE3-11 (SEQ ID NO: 15) (SEQ ID NO: 16) (SEQ ID NO: 17) 2E8Human KSSQSLLYSNGKTYLN VVSKLDS VQGTHYPFT AβpE3-11 (SEQ ID NO: 21)(SEQ ID NO: 22) (SEQ ID NO: 23) Difference KSSQSLLXSNGXXYLN XVSKLDSVQGTHXPFT (SEQ ID NO: 47) (SEQ ID NO: 48) (SEQ ID NO: 49) 1G11 MurineGLTLSDAWMN EIRSKAYKHATYYAESVKG HGS AβpE3-11 (SEQ ID NO: 32)(SEQ ID NO: 33) (SEQ ID NO: 34) 2E4 Murine GITLNDAWMTEIRNKANNHATNYAESVKG HSY AβpE3-11 (SEQ ID NO: 38) (SEQ ID NO: 39)(SEQ ID NO: 40) Difference GXTLXDAWMX EIRXKAXXHATXYAESVKG HXX(SEQ ID NO: 50) (SEQ ID NO: 51) (SEQ ID NO: 52) 1G11 Murine RASQGISSKMGHGTKLED VQYAQFPYT AβpE3-11 (SEQ ID NO: 35) (SEQ ID NO: 36)(SEQ ID NO: 37) 2E4 Murine HASQGIRNNIG HGTKLED VQYDQFPYT AβpE3-11(SEQ ID NO: 41) (SEQ ID NO: 42) (SEQ ID NO: 43) Difference XASQGIXXXXGHGTKLED VQYXQFPYT (SEQ ID NO: 53) (SEQ ID NO: 36) (SEQ ID NO: 55)

These conserved amino acids which are to be found between the antibodiesgenerated against AβpE3 must account for particular amino acids that areimportant for recognising the AβpE3.

The invention thus relates to an antibody, in particular an isolatedantibody, or a fragment thereof, specific for human AβpE3 which antibodycomprises, or consists, of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising        GYTFTDYYX₁N (SEQ ID NO:56),    -   a heavy chain variable region (V_(H)) CDR2 comprising        WX₂X₃PGSGNX₄KYNEKFKG (SEQ ID NO:57),    -   a heavy chain variable region (V_(H)) CDR3 comprising EGX₅X₆X₇Y        (SEQ ID NO:58),    -   a light chain variable region (V_(L)) CDR1 comprising        KSSQSLLX₈SNGX₉X₁₀YLN (SEQ ID NO:59),    -   a light chain variable region (V_(L)) CDR2 comprising X₁₁VSKLDS        (SEQ ID NO:60), and    -   a light chain variable region (V_(L)) CDR3 comprising        VQGTHX₁₂PFT (SEQ ID NO:61);    -   wherein X₁ to X₁₂ symbolizes a natural amino acid.

In a further embodiment X₁ to X₁₂ may be selected as follows

X₁ is I or L,

X₂ is I or L, X₃ is Y or N and/or X₄ is V or T,

X₅ is I or L, X₆ is I or P and/or X₇ is V or D,

X₈ is H or Y, X₉ is E or K and/or X₁₀ is T or S,

X₁₁ is A or V, and/or

X₁₂ is Y or F.

The invention thus also relates to an antibody, in particular anisolated antibody, or a fragment thereof, specific for murine AβpE3which antibody comprises, or consists, of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising        GX₁TLX₂DAWMX₃ (SEQ ID NO:62),    -   a heavy chain variable region (V_(H)) CDR2 comprising        EIRX₄KAX₅X₆HATX₇YAESVKG (SEQ ID NO:63),    -   a heavy chain variable region (V_(H)) CDR3 comprising HX₈X₉ (SEQ        ID NO:64),    -   a light chain variable region (V_(L)) CDR1 comprising        X₁₀ASQGIX₁₁X₁₂X₁₃X₁₄G (SEQ ID NO:65),    -   a light chain variable region (V_(L)) CDR2 comprising HGTKLED        (SEQ ID NO:36), and    -   a light chain variable region (V_(L)) CDR3 comprising        VQYX₁₅QFPYT (SEQ ID NO:66)    -   wherein X₁ to X₁₅ symbolizes a natural amino acid.

In a further embodiment X₁ to X₁₅ may be selected as follows

X₁ is I or L, X₂ is S or N, and/or X₃ is N or T,

X₄ is S or N, X₅ is Y or N, X₆ is K or N, and/or X₇ is Y or N,

X₈ is G or S and or S X₉ is S or Y,

X₁₀ is R or H, X₁₁ is S or R, X₁₂ is S or N, X₁₃ is K or N, and/or X₁₄is M or I, and/or

X₁₅ is A or D.

The antibody may be isolated and preferably be specific for human AβpE3with a binding affinity (K_(D)) of about 100 nM to about 50 nM, or about50 nM to about 10 nM, or about 10 nM to about 5 nM for the humanpyroglutamated Aβ fragment pEFRHDSGYE (SEQ ID NO:67) or the murinefragment pEFGHDSGFE (SEQ ID NO:68).

The invention further relates to an in vitro method for making anantibody, or a fragment thereof, specific for human pyroglutamated Aβfragment pEFRHDSGYE (SEQ ID NO:67) or the murine fragment pEFGHDSGFE(SEQ ID NO:68), which method comprises the step varying one or more ofthe amino acids X. X may be selected as outlined above. Any suitablemethod for constructing these fragments can be used, such as PCR, andsequencing followed by a screening and selection for antibodies with theabove mentioned desired properties with regards to K_(D) and affinityfor AβpE3-11 as shown in the examples.

The present invention provides the antibodies of the invention producedby a host cell. In one embodiment, the invention provides a monoclonalantibody produced by clone 5C9 or 2E8. In another embodiment, theinvention provides a monoclonal antibody produced by clone 2E4 or 1G11.In other embodiments, the invention provides an isolated antibody, orfragment thereof, that competes with binding with the monoclonalantibody produced by clone 5C9, 2E8, 2E4 or 1G11. Furthermore, theinvention provides hybridoma cell line 5C9, 2E8, 2E4 or 1G11, or progenythereof.

Accordingly the invention relates to an antibody, or a fragment thereof,comprising of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising SEQ ID        NO:12;    -   a heavy chain variable region (V_(H)) CDR2 comprising SEQ ID        NO:13;    -   a heavy chain variable region (V_(H)) CDR3 comprising SEQ ID        NO:14;    -   a light chain variable region (V_(L)) CDR1 comprising SEQ ID        NO:15;    -   a light chain variable region (V_(L)) CDR2 comprising SEQ ID        NO:16; and    -   a light chain variable region (V_(L)) CDR3 comprising SEQ ID        NO:17.

The antibody, or fragment thereof, may also comprise or consist of aheavy chain variable region (V_(H)) encoded by the nucleotide sequenceof SEQ NO:4 or having the amino acid sequence of SEQ ID NO:5. Theantibody, or fragment thereof, may further comprise or consist of alight chain variable region (V_(L)) encoded by the nucleotide sequenceof SEQ NO:6 or having the amino acid sequence of SEQ ID NO:7.Alternatively, the antibody, or fragment thereof, may comprise orconsist of the heavy chain variable region (V_(H)) and the light chainvariable region (V_(L)).

In another embodiment the antibody, or a fragment thereof, comprises orconsist of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising SEQ ID        NO:18;    -   a heavy chain variable region (V_(H)) CDR2 comprising SEQ ID        NO:19;    -   a heavy chain variable region (V_(H)) CDR3 comprising SEQ ID        NO:20;    -   a light chain variable region (V_(L)) CDR1 comprising SEQ ID        NO:21;    -   a light chain variable region (V_(L)) CDR2 comprising SEQ ID        NO:22; and    -   a light chain variable region (V_(L)) CDR3 comprising SEQ ID        NO:23.

The antibody, or fragment thereof, may comprise or consist of a heavychain variable region (V_(H)) encoded by the nucleotide sequence of SEQNO:8 or having the amino acid sequence of SEQ ID NO:9. The antibody, orfragment thereof, may further comprise or consist of a light chainvariable region (V_(L)) encoded by the nucleotide sequence of SEQ NO:10or having the amino acid sequence of SEQ ID NO:11. Alternatively, theantibody, or fragment thereof, may comprise or consist of the heavychain variable region (V_(H)) and the light chain variable region(V_(L)).

In a further embodiment the antibody, or a fragment thereof, maycomprise or consist of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising SEQ ID        NO:32;    -   a heavy chain variable region (V_(H)) CDR2 comprising SEQ ID        NO:33;    -   a heavy chain variable region (V_(H)) CDR3 comprising SEQ ID        NO:34;    -   a light chain variable region (V_(L)) CDR1 comprising SEQ ID        NO:35;    -   a light chain variable region (V_(L)) CDR2 comprising SEQ ID        NO:36; and    -   a light chain variable region (V_(L)) CDR3 comprising SEQ ID        NO:37.

The antibody, or fragment thereof, may comprise or consist of a heavychain variable region (V_(H)) encoded by the nucleotide sequence of SEQNO:24 or having the amino acid sequence of SEQ ID NO:25. The antibody,or fragment thereof, may further comprise or consist of a light chainvariable region (V_(L)) encoded by the nucleotide sequence of SEQ NO:26or having the amino acid sequence of SEQ ID NO:27. Alternatively, theantibody, or fragment thereof, may comprise or consist of the heavychain variable region (V_(H)) and the light chain variable region(V_(L)).

In a still further embodiment the invention relates to an antibody, or afragment thereof, comprising or consisting of:

-   -   a heavy chain variable region (V_(H)) CDR1 comprising SEQ ID        NO:38;    -   a heavy chain variable region (V_(H)) CDR2 comprising SEQ ID        NO:39;    -   a heavy chain variable region (V_(H)) CDR3 comprising SEQ ID        NO:40;    -   a light chain variable region (V_(L)) CDR1 comprising SEQ ID        NO:41;    -   a light chain variable region (V_(L)) CDR2 comprising SEQ ID        NO:42; and    -   a light chain variable region (V_(L)) CDR3 comprising SEQ ID        NO:43.

The antibody, or fragment thereof, may comprise or consist of a heavychain variable region (V_(H)) encoded by the nucleotide sequence of SEQNO:28 or having the amino acid sequence of SEQ ID NO:29. The antibody,or fragment thereof, may further comprise or consist of a light chainvariable region (V_(L)) encoded by the nucleotide sequence of SEQ NO:30or having the amino acid sequence of SEQ ID NO:31. Alternatively, theantibody, or fragment thereof, may comprise or consist of the heavychain variable region (V_(H)) and the light chain variable region(V_(L)).

The antibodies mentioned above may, according to one embodiment, furtherhave no more than 4 amino acid differences, or no more than 3 amino aciddifferences, or no more than 2 amino acid differences, or no more than 1amino acid difference from said CDR1-3 (V_(H) and/or V_(L)) sequences.

It is envisaged that the antibody, or fragment thereof, may behumanized, chimeric, or single-chain antibody.

Further the antibodies may be in a composition together with apharmaceutically acceptable carrier, e.g. for use in the treatment ofAlzheimer disease.

The invention also relates to a method of treating Alzheimer's Diseasein a patient, comprising administering to the patient in need of suchtreatment, a therapeutically effective amount of an antibody of theinvention or a composition as mentioned above

The antibodies may further be used in a diagnostic method or as adiagnostic imagining ligand.

The fragments, or immunogenic fragments, consisting of the AβpE3-11(pEFRHDSGYE) (SEQ ID NO:67) fragment and/or AβpE3-11-C (pE-FRHDSGYEC)(SEQ ID NO:2) fragment may be used for generating antibodies having abinding affinity (K_(D)) to such fragment of with a binding affinity(K_(D)) of about 100 nM to about 50 nM, or about 50 nM to about 10 nM,or about 10 nM to about 5 nM.

The invention further relates to a therapeutic composition consisting orcomprising of an AβpE3-11 (pEFRHDSGYE) (SEQ ID NO:67) fragment and/orthe AβpE3-11-C (pE-FRHDSGYEC) (SEQ ID NO:2) fragment in an amountsufficient to elicit the production of antibodies, or an antibody thatspecifically binds to such fragment, and a pharmaceutically acceptableadjuvant or carrier.

The therapeutic composition may comprise one or more antibodies thatbinds to AβpE3-11 (pEFRHDSGYE) (SEQ ID NO:67) fragment and/or AβpE3-11-C(pE-FRHDSGYEC) (SEQ ID NO:2) fragment with a binding affinity (K_(D)) tosuch fragment of about with a binding affinity (K_(D)) of about 100 nMto about 50 nM, or about 50 nM to about 10 nM, or about 10 nM to about 5nM.

The present invention provides the peptide AβpE3-11-C (pEFRHDSGYEC) (SEQID NO:2). In one embodiment, the present invention provides the peptideAβpE3-11-C dimer (pEFRHDSGYEC-CEYGSDHRFEp) (composed of two copies ofSEQ ID NO:2 linked C-terminus to C-terminus). In another embodiment, thepresent invention provides polypeptide AβpE3-11-C-keyhole limpethemocyanin: pEFRHDSGYEC-[KLH] (SEQ ID NO:2). The peptide AβpE3-11-C(pEFRHDSGYEC) (SEQ ID NO:2) conjugated to a carboxy-terminal carrier.

The present invention provides a method of immunizing an animalcomprising administering AβpE3-11-C (pEFRHDSGYEC) (SEQ ID NO:2) as animmunogen.

The present invention provides a therapeutic composition comprising theAβpE3-11 (pEFRHDSGYE) (SEQ ID NO:67) and/or the AβpE3-11-C (pEFRHDSGYEC)(SEQ ID NO:2) fragment in an amount sufficient to elicit the productionof antibodies, or an antibody that immunospecifically binds to suchfragment.

The invention also relates to an in vitro method for making an antibodyof, or a fragment thereof, specific for human pyroglutamated Aβ fragmentpEFRHDSGYE (SEQ ID NO:67) and/or pE-FRHDSGYEC (SEQ ID NO:2) or specificfor murine pyroglutamated Aβ fragment pEFGHDSGFE (SEQ ID NO:68) and/orpEFGHDSGFEC (SEQ ID NO:3), which method comprises the step varying oneor more of the amino acids “X” given above. In particular the in vitromethod the in vitro method may be suitable for making antibodies whichbinds with an affinity (KD) to said fragment of about 100 nM to about 50nM, or about 50 nM to about 10 nM, or about 10 nM to about 5 nM

The present invention relates to improvements in therapy and preventionof Alzheimer's disease (AD) and other diseases characterized bydeposition of amyloid, e. g. characterized by amyloid deposits in thecentral nervous system (CNS).

The present invention also provides a method of reducing Aβ plaqueformation in a patient, comprising administering to the patient in needof such treatment, a therapeutically effective amount of an antibody ofthe invention. In one embodiment, the Aβ plaques comprise pyroglutamatedAβ.

In one embodiment, the present invention provides a compositioncomprising the antibody described herein and a pharmaceuticallyacceptable carrier. In some embodiments, the composition of theinvention is provided for use in the treatment of Alzheimer disease, orin the treatment of a neurodegenerative or cognitive disease ordisorder.

The present invention also provides a method of treating Alzheimer'sDisease in a patient, comprising administering to the patient in need ofsuch treatment, a therapeutically effective amount of an antibody of theinvention. The present invention also provides a method of treatingneurodegenerative or cognitive disease or disorder in a patient,comprising administering to the patient in need of such treatment, atherapeutically effective amount of an antibody of the invention.

There are important parallels between AD and other neurologicaldiseases, including prion diseases (such as kuru, Creutzfeld-Jacobdisease and bovine spongiform encephalitis), Parkinson's disease,Huntington's disease, and fronto-temporal dementia. All involve depositsof abnormal proteins in the brain. AD and prion diseases cause dementiaand death, and both are associated with the formation of insolubleamyloid fibrils, but from membrane proteins that are different from eachother.

Monoclonal antibodies of the present invention may e.g. be produced bythe hybridoma method first described by Kohler et al., Nature 256, 495(1975), or may be produced by recombinant DNA methods. Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in, for example, Clackson et al., Nature 352,624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991).Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B lymphocyte cells obtained from miceimmunized with an antigen of interest, for instance, in the form ofcells expressing the antigen on the surface, or a nucleic acid encodingan antigen of interest. Monoclonal antibodies may also be obtained fromhybridomas derived from antibody-expressing cells of immunized humans ornon-human mammals such as rats, rabbits, dogs, primates, etc.

In one embodiment, the antibody of the invention is a human antibody.Human monoclonal antibodies directed against Aβ may be generated usingtransgenic or transchromosomal mice carrying parts of the human immunesystem rather than the mouse system. Such transgenic andtranschromosomic mice include mice referred to herein as HuMAb mice andKM mice, respectively, and are collectively referred to herein as“transgenic mice”.

The HuMAb mouse contains a human immunoglobulin gene miniloci thatencodes unrearranged human heavy variable and constant (μ and Y) andlight variable and constant (K) chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous p and K chainloci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, themice exhibit reduced expression of mouse IgM or K and in response toimmunization, the introduced human heavy and light chain transgenes,undergo class switching and somatic mutation to generate high affinityhuman IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113,49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764536-546 (1995)). The preparation of HuMAb mice is described in detail inTaylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J.et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J.Immunol. 152, 2912-2920 (1994), Taylor, L. et al., InternationalImmunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825,5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318,5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227,WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCol2 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCol2 human heavy chain transgene (as described in Example 2 of WO01/14424).

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (5C20) as described in WO02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according to wellknown techniques. Human monoclonal or polyclonal antibodies of thepresent invention, or antibodies of the present invention originatingfrom other species may also be generated transgenically through thegeneration of another non-human mammal or plant that is transgenic forthe immunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172and 5,741,957.

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be generated throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, and other techniques, usingtechniques well known in the art and the resulting molecules may besubjected to additional maturation, such as affinity maturation, as suchtechniques are well known in the art (see for instance Hoogenboom etal., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al.,Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNASUSA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992),Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. AcidsResearch 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130,43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S.Pat. No. 5,733,743). If display technologies are utilized to produceantibodies that are not human, such antibodies may be humanized.

The antibody of the invention may be of any isotype. The choice ofisotype typically will be guided by the desired effector functions, suchas ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4.Either of the human light chain constant regions, kappa or lambda, maybe used. If desired, the class of an anti-AβpE3 antibody of the presentinvention may be switched by known methods. For example, an antibody ofthe present invention that was originally IgM may be class switched toan IgG antibody of the present invention. Further, class switchingtechniques may be used to convert one IgG subclass to another, forinstance from IgG1 to IgG2. Thus, the effector function of theantibodies of the present invention may be changed by isotype switchingto, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody forvarious therapeutic uses. In one embodiment an antibody of the presentinvention is an IgG1 antibody, for instance an IgG1, κ.

In one embodiment, the antibody of the invention is a full-lengthantibody, preferably an IgG antibody, in particular an IgG1, κ antibody.In another embodiment, the antibody of the invention is an antibodyfragment or a single-chain antibody.

Antibodies fragments may e.g. be obtained by fragmentation usingconventional techniques, and the fragments screened for utility in thesame manner as described herein for whole antibodies. For example,F(ab′)₂ fragments may be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment may be treated to reduce disulfide bridges toproduce Fab′ fragments. Fab fragments may be obtained by treating an IgGantibody with papain; Fab′ fragments may be obtained with pepsindigestion of IgG antibody. An F(ab′) fragment may also be produced bybinding Fab′ described below via a thioether bond or a disulfide bond. AFab′ fragment is an antibody fragment obtained by cutting a disulfidebond of the hinge region of the F(ab′)₂. A Fab′ fragment may be obtainedby treating an F(ab′)₂ fragment with a reducing agent, such asdithiothreitol. Antibody fragment may also be generated by expression ofnucleic acids encoding such fragments in recombinant cells (see forinstance Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). Forexample, a chimeric gene encoding a portion of an F(ab′)₂ fragment couldinclude DNA sequences encoding the C_(H)1 domain and hinge region of theH chain, followed by a translational stop codon to yield such atruncated antibody fragment molecule.

In one embodiment, anti-AβpE3 antibody is a monovalent antibody,preferably a monovalent antibody as described in WO2007059782 (which isincorporated herein by reference in its entirety) having a deletion ofthe hinge region. Accordingly, in one embodiment, the antibody is amonovalent antibody, wherein said anti-pE3Aβ antibody is constructed bya method comprising:

-   -   (i) providing a nucleic acid construct encoding the light chain        of said monovalent antibody, said construct comprising a        nucleotide sequence encoding the V_(L) region of a selected        antigen specific anti-AβpE3 antibody and a nucleotide sequence        encoding the constant C_(L) region of an Ig, wherein said        nucleotide sequence encoding the V_(L) region of a selected        antigen specific antibody and said nucleotide sequence encoding        the C_(L) region of an Ig are operably linked together, and        wherein, in case of an IgG1 subtype, the nucleotide sequence        encoding the C_(L) region has been modified such that the C_(L)        region does not contain any amino acids capable of forming        disulfide bonds or covalent bonds with other peptides comprising        an identical amino acid sequence of the C_(L) region in the        presence of polyclonal human IgG or when administered to an        animal or human being;    -   (ii) providing a nucleic acid construct encoding the heavy chain        of said monovalent antibody, said construct comprising a        nucleotide sequence encoding the V_(H) region of a selected        antigen specific antibody and a nucleotide sequence encoding a        constant C_(H) region of a human Ig, wherein the nucleotide        sequence encoding the C_(H) region has been modified such that        the region corresponding to the hinge region and, as required by        the Ig subtype, other regions of the C_(H) region, such as the        C_(H)3 region, does not comprise any amino acid residues which        participate in the formation of disulphide bonds or covalent or        stable non-covalent inter-heavy chain bonds with other peptides        comprising an identical amino acid sequence of the C_(H) region        of the human Ig in the presence of polyclonal human IgG or when        administered to an animal human being, wherein said nucleotide        sequence encoding the V_(H) region of a selected antigen        specific antibody and said nucleotide sequence encoding the        C_(H) region of said Ig are operably linked together;    -   (iii) providing a cell expression system for producing said        monovalent antibody; and    -   (iv) producing said monovalent antibody by co-expressing the        nucleic acid constructs of (i) and (ii) in cells of the cell        expression system of (iii).

Similarly, in one embodiment, the anti-AβpE3 antibody is a monovalentantibody, which comprises:

-   -   (i) a variable region of an antibody of the invention as        described herein or an antigen binding part of the said region,        and    -   (ii) a C_(H) region of an immunoglobulin or a fragment thereof        comprising the C_(H)2 and C_(H)3 regions, wherein the C_(H)        region or fragment thereof has been modified such that the        region corresponding to the hinge region and, if the        immunoglobulin is not an IgG4 subtype, other regions of the        C_(H) region, such as the C_(H)3 region, do not comprise any        amino acid residues, which are capable of forming disulfide        bonds with an identical C_(H) region or other covalent or stable        non-covalent inter-heavy chain bonds with an identical C_(H)        region in the presence of polyclonal human IgG.

In a further embodiment, the heavy chain of the monovalent anti-AβpE3antibody has been modified such that the entire hinge has been deleted.

In another further embodiment, the sequence of said monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

Anti-AβpE3 antibodies of the invention also include single chainantibodies. Single chain antibodies are peptides in which the heavy andlight chain Fv regions are connected. In one embodiment, the presentinvention provides a single-chain Fv (scFv) wherein the heavy and lightchains in the Fv of an anti-AβpE3 antibody of the present invention arejoined with a flexible peptide linker (typically of about 10, 12, 15 ormore amino acid residues) in a single peptide chain. Methods ofproducing such antibodies are described in for instance U.S. Pat. No.4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNASUSA 85, 5879-5883 (1988) and McCafferty et al., Nature 348, 552-554(1990). The single chain antibody may be monovalent, if only a singleV_(H) and V_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used.

In general, anti-AβpE3 antibodies described herein may be modified byinclusion of any suitable number of such modified amino acids and/orassociations with such conjugated substituents. Suitability in thiscontext is generally determined by the ability to at least substantiallyretain AβpE3 selectivity and/or specificity associated with thenon-derivatized parent anti-AβpE3 antibody. The inclusion of one or moremodified amino acids may be advantageous in, for example, increasingpolypeptide serum half-life, reducing polypeptide antigenicity, orincreasing polypeptide storage stability. Amino acid(s) are modified,for example, co-translationally or post-translationally duringrecombinant production (e.g., N-linked glycosylation at N-X-S/T motifsduring expression in mammalian cells) or modified by synthetic means.Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenylated (e. g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEGylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998) Protein Protocols On CD-Rom, Humana Press, Totowa, N.J.The modified amino acid may, for instance, be selected from aglycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, or an amino acid conjugated to an organicderivatizing agent.

Anti-AβpE3 antibodies may also be chemically modified by covalentconjugation to a polymer to for instance increase their circulatinghalf-life. Exemplary polymers, and methods to attach them to peptides,are illustrated in for instance U.S. Pat. Nos. 4,766,106, 4,179,337,4,495,285 and 4,609,546. Additional illustrative polymers includepolyoxyethylated polyols and polyethylene glycol (PEG) (e.g., a PEG witha molecular weight of between about 1,000 and about 40,000, such asbetween about 2,000 and about 20,000, e.g., about 3,000-12,000 g/mol).

In one embodiment, anti-AβpE3 antibodies comprising one or moreradiolabeled amino acids are provided. A radiolabeled anti-AβpE3antibody may be used for both diagnostic and therapeutic purposes(conjugation to radiolabeled molecules is another possible feature).Non-limiting examples of labels for polypeptides include, but are notlimited to 3H, 14C, 15N, 35S, 90Y, 99Tc, and 1251, 1311, and 186Re.Methods for preparing radiolabeled amino acids and related peptidederivatives are known in the art (see for instance Junghans et al., inCancer Chemotherapy and Biotherapy 655-686 (2nd edition, Chafner andLongo, eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581,4,735,210, 5,101,827, 5,102,990 (U.S. RE35,500), U.S. Pat. Nos.5,648,471 and 5,697,902. For example, a radioisotope may be conjugatedby a chloramine T method.

In a further aspect, the invention relates to an expression vectorencoding an antibody of the invention. Such expression vectors may beused for recombinant production of antibodies of the invention.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-AβpE3 antibody-encoding nucleic acid is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in, for instance, Sykes and Johnston, Nat Biotech 12, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in, for instance, Schakowski et al., Mol Ther3, 793-800 (2001)), or as a precipitated nucleic acid vector construct,such as a CaP0₄-precipitated construct (as described in, for instance,WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigleret al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic CellGenetics 2, 603 (1981)). Such nucleic acid vectors and the usage thereofare well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and5,973,972).

In one embodiment, the vector is suitable for expression of anti-AβpE3antibody in a bacterial cell. Examples of such vectors includeexpression vectors such as BlueScript (Stratagene), pIN vectors (VanHeeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vectors(Novagen, Madison, Wis.) and the like).

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), and Grant et al., Methods in Enzymol 153,516-544 (1987)).

In an expression vector of the invention, anti-AβpE3 antibody-encodingnucleic acids may comprise or be associated with any suitable promoter,enhancer, and other expression-facilitating elements. Examples of suchelements include strong expression promoters (e. g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody of the invention as defined herein or a bispecificmolecule of the invention as defined herein. Examples of host cellsinclude yeast, bacterial, and mammalian cells, such as CHO or HEK cells.For example, in one embodiment, the present invention provides a cellcomprising a nucleic acid stably integrated into the cellular genomethat comprises a sequence coding for expression of an anti-AβpE3antibody of the present invention. In another embodiment, the presentinvention provides a cell comprising a non-integrated nucleic acid, suchas a plasmid, cosmid, phagemid, or linear expression element, whichcomprises a sequence coding for expression of an anti-AβpE3 antibody ofthe invention.

In a further aspect, the invention relates to a method for producing ananti-AβpE3 antibody of the invention, said method comprising the stepsof a) culturing a hybridoma or a host cell of the invention as describedherein above, and b) purifying the antibody of the invention from theculture media.

In an even further aspect, the invention relates to a pharmaceuticalcomposition comprising:

-   -   (i) an anti-AβpE3 antibody as defined herein or a bispecific        molecule as defined herein, and    -   (ii) a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 21thEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2005.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosencompound of the present invention and the chosen mode of administration.Suitability for carriers and other components of pharmaceuticalcompositions is determined based on the lack of significant negativeimpact on the desired biological properties of the chosen compound orpharmaceutical composition of the present invention (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.)) on antigen binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present invention employed, or the amidethereof, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe a aqueous or nonaqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the active compound in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. Parenteral compositions may be formulated in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the subjects to be treated; each unit contains apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

The effective dosages and the dosage regimens for the anti-AβpE3antibodies depend on the disease or condition to be treated and may bedetermined by the persons skilled in the art. An exemplary, non-limitingrange for a therapeutically effective amount of an antibody of thepresent invention is about 0.1-10 mg/kg/body weight, such as about 0.1-5mg/kg/body weight, for example about 0.1-2 mg/kg/body weight, such asabout 0.1-1 mg/kg/body weight, for instance about 0.15, about 0.2, about0.5, about 1, about 1.5 or about 2 mg/kg/body weight.

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the anti-AβpE3 antibody employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the present invention will be that amount of the compoundwhich is the lowest dose effective to produce a therapeutic effect. Suchan effective dose will generally depend upon the factors describedabove. Administration may e.g. be intravenous, intramuscular,intraperitoneal, or subcutaneous, and for instance administered proximalto the site of the target. If desired, the effective daily dose of apharmaceutical composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalcomposition as described above.

Labeled antibodies of the invention can be used for diagnostic purposesto detect, diagnose, or monitor diseases or disorders. The inventionprovides for the detection or diagnosis of a neurodegenerative orcognitive disease or disorder, including but not limited to Alzheimer'sDisease, comprising: (a) assaying the existence of pyroglutamated Aβfragments in cells or tissue samples of a subject using one or moreantibodies that immunospecifically bind to AβpE3; and (b) comparing thelevel of the antigen with a control level, e.g. levels in normal tissuesamples, whereby an increase in the assayed level of antigen compared tothe control level of antigen is indicative of the disease or disorder,or indicative of the severity of the disease or disorder.

Antibodies of the invention can be use to assay pyroglutamated Aβfragments in a biological sample using immunohistochemical methodswell-known in the art. Other antibody-based methods useful for detectingprotein include immunoassays such as the enzyme linked immunoassay(ELISA) and the radioimmunoassay (RIA). Suitable antibody labels may beused in such kits and methods, and labels known in the art includeenzyme labels, such as alkaline phosphatase and glucose oxidase;radioisotope labels, such as iodine (¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹²¹In), and technetium (^(99m)Tc); andluminescent labels, such as luminol and luciferase; and fluorescentlabels, such as flourescein and rhodamine.

Presence of labeled antibodies may be detected in vivo for diagnosispurposes. In one embodiment, diagnosis comprises: a) administering to asubject an effective amount of a labeled antibody; b) waiting for a timeinterval following administration for permitting labeled antibody toconcentrate at sites where Aβ may be detected and to allow for unboundlabeled antibody to be cleared to background level; c) determining abackground level; and d) detecting the labeled antibody in the subject,such that detection of labeled antibody above the background level isindicative that the subject has the disease or disorder, or isindicative of the severity of the disease or disorder. In accordancewith such embodiment, the antibody is labeled with an imaging moietysuitable for detection using a particular imaging system known to thoseskilled in the art. Background levels may be determined by variousmethods known in the art, including comparing the amount of labeledantibody detected to a standard value previously determined for aparticular imaging system. Methods and systems that may be used in thediagnostic methods of the invention include, but are not limited to,computed tomography (CT), whole body scan such as positron emissiontomography (PET), magnetic resonance imaging (MRI), and sonography.

EXAMPLES Example 1: Immunization and Screening

Pyroglutamated human Aβ3-11, with the addition of a C-terminal cysteineresidue, pE-FRHDSGYE-C (SEQ ID NO:2) and pyroglutamated murine Aβ3-11,with the addition of a C-terminal cysteine residue, pE-FGHDSGFE-C (SEQID NO:3) was used in the immunisations. As such, mice were immunizedwith pE-FRHDSGYE-C or with pEFRHDSGYE-C each conjugated to KLH. Thepeptides were made and amplified using PCR and purified as disclosed inSambrook (2000) Molecular Cloning, third edition, Cold Spring HarborLaboratory Press

The modified peptide was conjugated to a carrier protein, such as KLH.10 Balb/c mice were immunized and boosted with a standard immunizationprotocol as outlined below

Scheduled Procedure days Routes and dosages 1^(st) Immunization 0 10-100ug/0.25 ml animal, CFA 2^(nd) Immunization 14 10-50 ug/0.25 ml animal,IFA 3^(rd) Immunization 35 10-50 ug/0.25 ml animal, IFA Finalimmunization 56 10-50 ug/0.25 ml animal, Saline,

Tail bleedings were screened by ELISA or other method if needed withpyroglutamate and non-pyroglutamate-peptide.

Fusion and Screening.

2 fusions (1-2 spleens per fusion) were performed on the best mice thatresponded the best to the pyroglutamate-peptide in Example 1.

The hybridoma cells were screened by ELISA or other method if neededwith pyroglutamate and non-pyroglutamate-peptide and all positive cloneswere expanded into 24-well plates. The clones with best ELISA value andspecificity to pyroglutamate-peptide were selected for subcloning.

Subcloning, Expansion, Cryopreservation and Delivery.

Hybridoma subclones were screened by ELISA with pyroglutamate andnon-pyroglutamate-peptide. Up to five positive parental clones bylimiting dilution, isotype the final positive supernatants, and expandand cryopreserve two subclones for each parental.

Antibodies were purified by protein A chromatography of media fromcultured hybridoma cell.

Example 2: Characterization of Monoclonal Antibodies

Affinity measurements using SPR technology: Binding affinity of anti-Aβantibodies was determined using BIAcore 3000 (Biacore, Upsula, Sweden).Human and murine pyroglutaminyl and unmodified Aβ peptides with aC-terminal Cystein were immobilized on a CM5 chip using the2-(2-pyridinyldithio) ethaneamine hydrochloride (PDEA) couplingtechnology (GE healthcare).

Approximately 100 RU of peptide was immobilised. Antibody binding wasdetermined at different concentrations and kinetic constant calculatedfrom the combined set of data using bivalent binding kinetics.Association and dissociation constants were determined usingBIAevaluation software (version 3.1).

The BIAcore binding is shown in FIG. 1A-1B.

Example 3: Immunohistochemistry Methods

Paraffin blocks of frontal cortex tissue from Alzheimer patients andfrom age-matched controls were obtained from Cambridge Bioscience (UK)and sectioned at 4 μm. Likewise, sections were prepared from brains ofAPP/PS1 double-transgenic mice carrying the APP Swedish mutation.Deparaffinised sections were subjected to antigen retrieval by microwavetreatment in 10 mM Citrate buffer, pH 6, followed by 88% formic acid for3 min. Sections were incubated o/n at 4° C. with primary antibodies:Anti-Abeta #6E10 at 1:100 (Covance, N.J., USA); pE3-Abeta antibodies(clone 2E8G6 and 5C9) at dilutions ranging from 3-20 μg/ml. This wasfollowed by biotin-conjugated anti-mouse antibodies (#0464, DAKO,Denmark) at 1:500 and StreptAvidin-Biotin complex (Vector Laboratoreis,UK). The immunoreaction was developed in 0.05% diaminobenzidine+0.01%H₂O₂.

Binding studies indicated that the anti-AβpE3 antibodies, generated fromthe AβpE3-11 immunogen, are highly specific to pyroglutamated pE3peptide. As such, the generated anti-AβpE3 antibodies bind with littleor no affinity to the unmodified Aβ 3-11 peptide.

Further, antibodies raised against the human peptide did not bind themurine pyroglutamated peptide, and vice versa (see FIG. 1A-1B).Comparison of the human and the murine 3-11 amino acid sequences of Aβreveals two amino acid residue differences at Aβ positions 4 and 10. Weobserve that the amino acid residue Y at position 10 in the humanpeptide may contribute to the higher degree of specificity seen byanti-human AβpE3 antibodies.

It was demonstrated by immunohistochemistry in brain samples fromAlzheimer patients as well as APP/PS1 transgenic mice that the twoanti-AβpE3 monoclonal antibodies 5C9 and 2E8 (which is another specificmonoclonal antibody not claimed in this application) were able to bindto Abeta plaques (FIG. 2A-2B and FIG. 3A-3B). Immunostaining using arecognised commercial anti-Abeta antibody (clone 6E10) was compared tothe anti-AβpE3 monoclonal antibodies 2E8 and 5C9. AβpE3 antibodies wereshown to label only a subset of plaques at the dilutions tested, whereasintracellular Abeta and diffuse plaques were also stained by the 6E10antibody. The intracellular Abeta and diffuse plaques were not detectedby AβpE3 antibodies. All 3 antibodies clearly stained dense-coreplaques. pE3-Abeta antibodies did not label structures in human cortexfrom non-demented controls.

Those skilled in the art will recognize that various changes and/ormodifications may be made to aspects or embodiments of this inventionand that such changes and/or modifications may be made without departingfrom the spirit of this invention. Therefore, it is intended that theappended claims cover all such equivalent variations as will fall withinthe spirit and scope of this invention.

Each reference cited in the present application, including literaturereferences, books, patents and patent applications, is incorporatedherein by reference in its entirety.

What is claimed is:
 1. An IgG antibody, or a fragment thereof, whereinsaid antibody and said fragment are capable of specifically bindingmurine AβpE3 with a binding affinity (K_(D)) of about 100 nM to about 5nm for the murine pyroglutamated Aβ fragment pEFGHDSGFE (SEQ ID NO:68),wherein said antibody comprises: (A) a heavy chain variable region(V_(H)) CDR1 comprising GLTLSDAWMN (SEQ ID NO:32) or GITLNDAWMT (SEQ IDNO:38); (B) a heavy chain variable region (V_(H)) CDR2 comprisingEIRSKAYKHATYYAESVKG (SEQ ID NO:33) or EIRNKANNHATNYAESVKG (SEQ IDNO:39); (C) a heavy chain variable region (V_(H)) CDR3 comprising NO:64)HGS (SEQ ID NO:34) or HSY (SEQ ID NO:40); (D) a light chain variableregion (V_(L)) CDR1 comprising RASQGISSKMG (SEQ ID NO:35) or HASQGIRNNIG(SEQ ID NO:41); (E) a light chain variable region (V_(L)) CDR2comprising HGTKLED (SEQ ID NO:36) or HGTKLED (SEQ ID NO:42); and (F) alight chain variable region (V_(L)) CDR3 comprising VQYAQFPYT (SEQ IDNO:37) or VQYDQFPYT (SEQ ID NO:43).
 2. The antibody, or said fragmentthereof, of claim 1, wherein: (A) said heavy chain variable region(V_(H)) CDR1 comprises GLTLSDAWMN (SEQ ID NO:32); (B) said heavy chainvariable region (V_(H)) CDR2 comprises EIRSKAYKHATYYAESVKG (SEQ IDNO:33); (C) said heavy chain variable region (V_(H)) CDR3 comprises HGS(SEQ ID NO:34); (D) said light chain variable region (V_(L)) CDR1comprises RASQGISSKMG (SEQ ID NO:35); (E) said light chain variableregion (V_(L)) CDR2 comprises HGTKLED (SEQ ID NO:36); and (F) said lightchain variable region (V_(L)) CDR3 comprises VQYAQFPYT (SEQ ID NO:37).3. The antibody, or said fragment thereof, of claim 2, wherein saidantibody comprises: (A) a heavy chain variable region (V_(H)) thatcomprises the amino acid sequence of SEQ ID NO:25; and/or (B) a lightchain variable region (V_(L)) that comprises the amino acid sequence ofSEQ ID NO:27.
 4. The antibody, or said fragment thereof, of claim 1,wherein: (A) said heavy chain variable region (V_(H)) CDR1 comprisesGITLNDAWMT (SEQ ID NO:38); (B) said heavy chain variable region (V_(H))CDR2 comprises EIRNKANNHATNYAESVKG (SEQ ID NO:39); (C) said heavy chainvariable region (V_(H)) CDR3 comprises HSY (SEQ ID NO:40); (D) saidlight chain variable region (V_(L)) CDR1 comprises HASQGIRNNIG (SEQ IDNO:41); (E) said light chain variable region (V_(L)) CDR2 comprisesHGTKLED (SEQ ID NO:42); and (F) said light chain variable region (V_(L))CDR3 comprises VQYDQFPYT (SEQ ID NO:43).
 5. The antibody, or saidfragment thereof, of claim 4, wherein said antibody comprises: (A) aheavy chain variable region (V_(H)) that comprises the amino acidsequence of SEQ ID NO:29; and/or (B) a light chain variable region(V_(L)) that comprises the amino acid sequence of SEQ ID NO:31.
 6. Theantibody, or said fragment thereof, of claim 1, wherein said antibody isa humanized antibody, a chimeric antibody, or a single-chain antibody.7. The antibody, or said fragment thereof, of claim 1, wherein saidantibody or fragment is detectably labelled.
 8. A pharmaceuticalcomposition comprising the antibody, or said fragment thereof, of claim1 and a pharmaceutically acceptable carrier.
 9. The pharmaceuticalcomposition of claim 8, wherein: (A) said heavy chain variable region(V_(H)) CDR1 comprises GLTLSDAWMN (SEQ ID NO:32); (B) said heavy chainvariable region (V_(H)) CDR2 comprises EIRSKAYKHATYYAESVKG (SEQ IDNO:33); (C) said heavy chain variable region (V_(H)) CDR3 comprises HGS(SEQ ID NO:34); (D) said light chain variable region (V_(L)) CDR1comprises RASQGISSKMG (SEQ ID NO:35); (E) said light chain variableregion (V_(L)) CDR2 comprises HGTKLED (SEQ ID NO:36); and (F) said lightchain variable region (V_(L)) CDR3 comprises VQYAQFPYT (SEQ ID NO:37).10. The pharmaceutical composition of claim 9, wherein said antibodycomprises: (A) a heavy chain variable region (V_(H)) that comprises theamino acid sequence of SEQ ID NO:25; and/or (B) a light chain variableregion (V_(L)) that comprises the amino acid sequence of SEQ ID NO:27.11. The pharmaceutical composition of claim 8, wherein: (A) said heavychain variable region (V_(H)) CDR1 comprises GITLNDAWMT (SEQ ID NO:38);(B) said heavy chain variable region (V_(H)) CDR2 comprisesEIRNKANNHATNYAESVKG (SEQ ID NO:39); (C) said heavy chain variable region(V_(H)) CDR3 comprises HSY (SEQ ID NO:40); (D) said light chain variableregion (V_(L)) CDR1 comprises HASQGIRNNIG (SEQ ID NO:41); (E) said lightchain variable region (V_(L)) CDR2 comprises HGTKLED (SEQ ID NO:42); and(F) said light chain variable region (V_(L)) CDR3 comprises VQYDQFPYT(SEQ ID NO:43).
 12. The pharmaceutical composition of claim 11, whereinsaid antibody comprises: (A) a heavy chain variable region (V_(H)) thatcomprises the amino acid sequence of SEQ ID NO:29; and/or (B) a lightchain variable region (V_(L)) that comprises the amino acid sequence ofSEQ ID NO:31.